In recent years considerable effort has been directed at applying gene delivery techniques. That term describes a wide variety of methods using recombinant biotechnology techniques to deliver a variety of different materials to a cell. These methods include, for example, vectors such as viral vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene gun, catheters, etc. The different techniques used depend in part upon the gene being transferred and the purpose therefore. Thus, for example, there are situations where only a short-term expression of the gene is desired in contrast to situations where a longer term, even permanent expression of the gene is desired.
Vectors that have been looked at include both DNA viral vectors and RNA viral vectors. For example, DNA vectors include pox vectors such as orthopox or avipox vectors (see, e.g., U.S. Pat. No. 5,656,465), herpes virus vectors, such as herpes simplex I Virus (HSV) vectors [Geller, A. I. et al., J. Neurochem. 64:487 (1995); Lim, F., et al., DNA Cloning: Mammalian Systems, D. Glover, Ed., Oxford Univ. Press, Oxford, England (1995); Geller, A. I. et al., Proc. Natl. Acad. Sci., U.S.A. 90:7603 (1993)]: Adenovirus vectors [Legal Lasalle et al., Sci. 259–988 (1993); Davidson et al., Nat. Genet. 3:219 (1993); Yang et al., J. Virol., 69:2004 (1995)]; and Adeno Associated Virus Vectors [Kaplitt, M. G., et al., Nat. Genet. 8;148 (1994)]. Retroviral vectors include Moloney murine leukemia viruses (MMLV) and human immunodeficiency viruses (HIV) [See, U.S. Pat. No. 5,665,577].
Recently, a great deal of attention has been focused on problems that may have arisen with clinical trials using gene delivery techniques. Many of the criticisms that have been raised do not address the value of such work, but rather failures to follow reporting procedures where adverse reaction and deaths occur. The individuals now receiving gene therapy are typically ill. Many have proved refractory to other treatment protocols. A number of these individuals have extremely poor life expectancies. Thus, as with other trials of patients in such poor health, problems can arise. It would be desirable to further improve gene delivery systems to reduce such problems.
Further, while attention has been focused on the use of viral vectors, particularly for in vivo therapy, for example, in somatic cell therapy or direct in vivo applications, many other applications exist such as in in vitro assays.
Various vectors have characteristics that make them desirable for certain applications. For example, a retroviral vector can be used to infect a host cell and have the genetic material integrated into that host cell with high efficiency. One example of such a vector is a modified Moloney murine leukemia virus (MMLV), which has had its packaging sequences deleted to prevent packaging of the entire retroviral genome. However, that retrovirus does not transduce resting cells. Additionally, since many retroviruses typically enter cells via specific receptors, if the specific receptors are not present on a cell or are not present in large enough numbers, the infection is either not possible or is inefficient. Concerns have also been expressed as a result of outbreaks of wild-type viruses from the recombinant MMLV producing cell lines, i.e., reversions.
An adenovirus vector can infect a wide range of cells. However, the transferred genetic material is not integrated. Therefore, it must be administered repeatedly. Additionally, most individuals have natural immunity to such vectors. Thus, high volumes of virus are needed when using the vector for gene delivery. The reason for this is the body's own immune system more readily attacks these vectors.
Recently, attention has focused on lentiviral vectors such as those based upon the primate lentiviruses, e.g., human immunodeficiency viruses (HIV) and simian immunodeficiency virus (SIV). HIV vectors can infect quiescent cells in addition to dividing cells. Moreover, by using a pseudotyped vector (i.e., one where an envelope protein from a different species is used), problems encountered with infecting a wide range of cell types can be overcome by selecting a particular envelope protein based upon the cell you want to infect. Additionally, in view of the complex gene splicing patterns seen in a lentiviruses such as HIV, multivalent vectors (i.e., those expressing multiple genes) having a lentiviral core, such as an HIV core, are expected to be more efficient. Yet, despite the advantages that HIV based vectors offer, there is still a concern with the use of HIV vectors in view of the severity of HIV infection. Thus, means for providing additional safeguards, such as attenuated forms that are less likely to revert to a wild type virus are desirable.
Variations can be made where multiple modifications are made, such as deleting nef. rev, vif and vpr genes. One can also have the 3′ and 5′ U3 deleted LTRs.
Marasco et al. discovered a method by which one could express antibodies within a cell and have them bind to a target within that cell. [See U.S. Pat. No. 5,851,829 to Marasco and Haseltine]. These intracellularly expressed antibodies (intrabodies) can be used in a method of functional genomics. In this manner, one can take a specific unknown gene, express its gene product, use that gene product to generate an antibody thereto and use the antibody intracellularly to “knock-out” the putative protein in the cell. Thereafter one can compare that cell to a control cell to determine the effect the loss of its gene product has on the cell in both in vitro and in vivo systems. This method requires generation of a specific antigen and antibody thereto. It would be desirable to have a method to take advantage of the efficiencies of this approach with large numbers of members of a particular group.
It would be highly desirable to have a vector and a method of use thereof where one could look for any molecule resulting in a particular function and rapidly determine that molecule, e.g. protein. It would be very desirable to be able to do this in an automated manner permitting rapid identification of the desired molecule.